Genetic basis of nectar guide trichome variation between bumblebee- and self-pollinated monkeyflowers (Mimulus): role of the MIXTA-like gene GUIDELESS

Nectar guide trichomes play crucial ecological roles in bee-pollinated flowers, as they serve as footholds and guides for foraging bees to access the floral rewards. However, the genetic basis of natural variation in nectar guide trichomes among species remains poorly understood. In this study, we performed genetic analysis of nectar guide trichome variation between two closely related monkeyflower (Mimulus) species, the bumblebee-pollinated Mimulus lewisii and self-pollinated M. parishii. We demonstrate that a MIXTA-like R2R3-MYB gene, GUIDELESS, is a major contributor to the nectar guide trichome length variation between the two species. The short-haired M. parishii carries a recessive allele due to non-synonymous substitutions in a highly conserved motif among MIXTA-like MYB proteins. Furthermore, our results suggest that besides GUIDELESS, additional loci encoding repressors of trichome elongation also contribute to the transition from bumblebee-pollination to selfing. Taken together, these results suggest that during a pollination syndrome switch, changes in seemingly complex traits such as nectar guide trichomes could have a relatively simple genetic basis, involving just a few genes of large effects. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-04736-y.

The monkeyflower species Mimulus lewisii and M. parishii are closely related [59,60] but display different pollination modes.The bumblebee-pollinated M. lewisii bears large showy flowers with long nectar guide trichomes (Fig. 1a, left panel), whereas the self-pollinated M. parishii produces small flowers with very short nectar guide trichomes (Fig. 1a, middle panel).These species are easy to grow in the greenhouse, with short generation time and high fecundity, and are amenable to transgenic manipulation through Agrobacterium-mediated transformation [61,62].This study represents the first step towards a detailed dissection of the genetic basis underlying nectar guide trichome variation between the two species.Starting from an F2 population with a wide range of nectar guide trichome lengths (Fig. 1b), we constructed near-isogenic lines (NILs) in the M. lewisii genetic background to reduce the complexity of F2 phenotypes into Mendelian loci.Through bulked segregant analysis, finescale genetic mapping, and complementation crosses, we demonstrate that the MIXTA-like gene, GUIDELESS [46], is a major contributor to the nectar guide trichome variation between the two species.

Plant materials and growth conditions
The M. lewisii inbred line LF10, the guideless mutant, and the M. parishii inbred line Mpar were described previously [46,61,62].All plants were grown in the University of Connecticut EEB research greenhouses under natural light supplemented with sodium vapor lamps, ensuring a 16-hr day length with a light intensity of 110-160 µmol•m − 2 •s − 1 .Plants were fertilized 2-3 times per week.

Quantification of nectar guide trichome length
Nectar guide tissues at the same site near the throat (marked by a red circle in Fig. 1a) of the corolla were cut into small pieces.The trichomes in these tissues were then imaged under a light microscope, and their lengths were measured using Zeiss ZEN 2.6 lite (blue edition) software.

NIL construction
To construct NILs with loci controlling nectar guide trichome variation introgressed from M. parishii to M. lewisii, we selected an F2 individual with short trichomes, ZH101 (Fig. 1b), to backcross (BC) to M. lewisii, and then we selfed one BC 1 individual that most closely resembled M. lewisii, including the long nectar guide trichomes.The short-haired phenotype reappeared in the selfing population (BC 1 S 1 ).The same process was repeated for a second time.The short vs. long trichome segregated ~ 1:3 in the BC 2 S 1 population.The short-haired individuals from the BC 2 S 1 population were then subjected to bulked segregant analysis by genome sequencing to identify the causal locus (see the section below).One of the short-haired BC 2 S 1 individuals was backcrossed to M. lewisii again to clean up the residual M. parishii DNA, and one shorthaired BC 3 S 1 individual with only the causal locus introgressed from M. parishii was retained as the G P/P NIL (named for the causal gene GUIDELESS; see Results).

Bulked segregant analysis by deep sequencing
To determine which chromosome fragment (s) was introgressed from M. parishii to M. lewisii in the G P/P NIL, we performed bulked segregant analysis, following [63].Briefly, we pooled DNA samples from 39 short-haired individuals from the ZH101 BC 2 S 1 population, with equal representation from each sample.A small-insert (350-bp) library was prepared for the pooled sample, and 150-bp paired-end reads were generated by Illumina NovaSeq at Novogene (Sacramento, CA), with ~ 80-fold genome coverage.The resulting short reads were mapped to the M. lewisii reference genome (http://mimubase.org/FTP/Genomes/LF10g_v2.0/) with CLC Genomics Workbench 7.0.After SNP calling, the number of homozygous SNPs in 20-kb bins was plotted in a bar graph.

Expression analysis using RT-qPCR
Total RNA was extracted using the Spectrum Plant Total RNA Kit (TRN250, Sigma-Aldrich) and cDNA was synthesized from 500 ng of the DNaseI (Invitrogen) treated RNA using GoScript™ Reverse Transcription Mix (A2791, Promega), then diluted 10-fold before RT-qPCR.To normalize expression levels, the M. lewisii/M.parishii ortholog of Arabidopsis ubiquitin-conjugating enzyme gene (At5g25760), MIUBC, was used as reference gene following [62].RT-qPCR was performed using iQ TM SYBR® Green Supermix (Bio-Rad) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad).The cDNA samples were amplified for 40 cycles of 95 °C for 15 s and 60 °C for 60 s.Amplification efficiencies for each primer pair were determined using critical threshold values obtained from a dilution series (1:4, 1:8, 1:16, 1:32) of pooled cDNAs.Relative expression of GUIDELESS was calculated using the formula (E target ) CP (ref ) / (E ref ) CP (target) .The primers used for RT-(q)PCR are listed in Table S1.

Variation in nectar guide trichome length between M. lewisii and M. parishii
Measurements using the microscope images showed that the nectar guide trichome length of M. lewisii is about 8 times that of M. parishii and about 3 times that of the F1 hybrid (Fig. 1c, d).We visually scored the nectar guide trichome lengths of 264 F2 individuals, and categorized them into four classes (examples shown in Fig. 1b).Class I (14.4%; 38/264) have very short trichomes resembling M. parishii.Class IV (8.3%; 22/264) showed long trichomes similar to M. lewisii.Class II (48.5%; 128/264) and class III (28.8%; 128/264) showed intermediate trichome lengths, with the former closer to M. parishii and the latter closer to M. lewisii (Fig. 1e).The frequency distribution of the F2 phenotypes suggests a non-monogenic basis of nectar guide trichome variation between the two species.

Identification of GUIDELESS as a candidate gene underlying one of the causal loci
To reduce the variation in nectar guide trichome length displayed by the F2 population into mendelian loci, we took a near-isogenic line (NIL) approach.Specifically, we selected the F2 individual ZH101 (Fig. 1b) bearing short nectar guide trichomes, and backcrossed it to M. lewisii.Within the resulting BC 2 S 1 population (two rounds of backcrossing and selfing; see Materials and Methods), approximately 3/4 (111/154) of the individuals had nectar guide trichomes similar to the wild type M. lewisii and ~ 1/4 (43/154) had shorter trichomes.We thus inferred that this short-haired NIL is homozygous for the recessive M. parishii allele at a single causal locus.
To identify the chromosomal location of this locus, we performed a bulked segregant analysis of pooled DNA samples from 39 short-haired individuals in the BC 2 S 1 population.Illumina sequencing of the bulked DNA revealed four relatively small genomic regions homozygous for M. parishii (Fig. 2a), one of which was expected to contain the causal gene.From the BC 2 S 1 population, we selected one short-haired individual that was most similar to M. lewisii and backcrossed it one more time to M. lewisii.Selfing one of the BC 3 individuals produced the BC 3 S 1 population.By fine-mapping 96 BC 3 S 1 individuals, we found that a 460-kb DNA segment on chromosome 6 (Chr 6: 1,533,002-1,988,221; between markers MLCP6_200 and MLCP6_250) co-segregated with nectar guide trichome length (Fig. 2b).This segment contains the GUIDELESS gene, which encodes a MIXTAlike R2R3-MYB transcription factor and was previously shown to control nectar guide trichome development in M. lewisii through mutant analysis [46].Therefore, GUIDELESS was considered the most promising candidate gene.Genotyping the BC 3 S 1 population also allowed us to identify a high-quality NIL that is homozygous for the M. parishii GUIDELESS allele in an otherwise M. lewisii genomic background, named the G P/P NIL (Fig. 2c).

Verification of GUIDELESS as the causal gene
To verify gene causality, we performed a complementation test by crossing the previously characterized lossof-function guideless mutant (in the M. lewisii LF10 background; Fig. 2c) with the G P/P NIL.If GUIDELESS is the causal gene underlying the short trichome phenotype of the G P/P NIL, we would expect that crossing the null guideless mutant with the G P/P NIL, which is homozygous for the recessive M. parishii allele, should result in F1 progeny (GN_F1) with short trichomes (i.e., the recessive G P allele and the null mutant allele do not complement one another).This was exactly what we observed: the F1 progeny (GN_F1) produced nectar guide trichomes of comparable length to those in the G P/P NIL (Fig. 2c, d).By contrast, the F1 progeny from the cross between the guideless mutant and wild-type M. lewisii (GL_F1) produced nectar guide trichomes comparable to the wild type in length (Fig. 2c, d).These results provide strong evidence that GUIDELESS is indeed the causal gene underlying the G P/P NIL phenotype.
The observation that nectar guide trichomes in the G P/P NIL are much shorter than those in the wild-type M. lewisii but still substantially longer than those in the null guideless mutant (Fig. 2d) indicates that the GUIDELESS allele in M. parishii is hypomorphic (i.e., partially functional).
Consistent with this inference, expression level of the MpGUIDELESS allele was only slightly lower than that of the MlGUIDELESS allele (Fig. 2e) and the MpGUIDE-LESS coding DNA does not contain obvious null mutations (e.g., premature stop codons).However, the predicted MpGUIDELESS protein sequence has two amino acid substitutions ("SA" to "TT") in a highly conserved motif among MIXTA-like R2R3-MYBs (Fig. 3), potentially attenuating protein function.

Other loci underlying nectar guide trichome length variation between M. lewisii and M. parishii
Our measurements showed that the GUIDELESS locus only accounts for ~ 40% of the parental difference in nectar guide trichome length between M. lewisii and M. parishii (Fig. 2d), implying the involvement of other loci underlying trichome length variation.Moreover, the fact that F1 hybrids between M. lewisii and M. parishii have much shorter nectar guide trichomes than M. lewisii (Fig. 1a, c, d), whereas the GUIDELESS NIL is similar to wild-type M. lewisii in a heterozygous state, suggests the existence of at least one other locus negatively regulating nectar guide trichome length, with the M. parishii allele dominant over the M. lewisii allele.In fact, the proportion of F2 individuals with very short trichomes (class I: 14.4%) is remarkably consistent with the expected ratio of a three-loci model.If the M. parishii allele is also dominant at the third locus, individuals with very short trichomes are expected to be homozygous for the M. parishii allele at GUIDELESS, and either homozygous for the M. parishii allele or heterozygous at the other two loci.That is, a ratio of 14.1% (1/4 * 3/4 * 3/4 = 9/64).

Discussion
In this study we analyzed the genetic basis of variation in nectar guide trichome length between two Mimulus species with distinct pollination syndromes.The two species differ by 8-fold in nectar guide trichome length.Our results suggest that the GUIDELESS gene is a major contributor to nectar guide trichome length variation between the bumblebee-pollinated M. lewisii and selfpollinated M. parishii.Compared to other pollinatorassociated floral traits such as coloration, nectar guide trichome variation has received little attention.Our study shows that in the M. lewisii-M.parishii system, nectar guide trichome is a typical "complex" trait, as reflected by the continuous variation in the F2 population, but can be dissected into just a few mendelian loci by constructing NILs.
The level of difficulty in generating high quality NILs (e.g., with introgressed chromosome fragments less than 500 kb) and ultimately identifying the causal genes depends on local recombination rates.The GUIDE-LESS locus has an exceptionally high recombination rate.Genotyping 96 ZH101_BC 3 S 1 individuals was sufficient to narrow down the causal gene to a 460-kb interval (Fig. 2b).The high recombination rate in this region is also consistent with previous bulked segregant analysis of the guideless mutant [46].Genome sequencing of 100 individuals from an intra-specific F2 population was enough to locate the causal mutation to a 50-kb interval.However, it is not unusual for certain loci to undergo low recombination in inter-specific crosses.For example, in our previous studies of flower color variation between M. lewisii and the closely related M. cardinalis, as well as between M. parishii and M. cardinalis, the generation of high quality NILs of flower color loci required genotyping of 1,000-3,000 individuals from the final mapping population [61][62][63][64].Generation of high quality NILs at the other inferred nectar guide trichome loci is currently underway.The anticipated NILs will enable us to investigate the genetic interactions among these loci and to ultimately identify the causal genes.
The most recent phylogenomic analysis of M. lewisii, M. parishii, and their close relatives [60] suggests that the self-pollination syndrome of M. parishii is a derived state, including not only short necatar guide trichomes, but also small flower size, inconspicuous coloration, and reduced anther-stigma separation.The inconspicuous coloration of M. parishii flowers was recently shown to be caused by a mutation in the 5' UTR of an anthocyanin-activating R2R3-MYB gene PELAN [61].This mutation does not alter protein function or gene transcript level.Instead, it inhibits protein translation, and therefore represents a type of loss-of-function mutation underlying the recessive allele in M. parishii.Although the specific molecular mechanisms are very different, the GUIDELESS allele in M. parishii is somewhat similar to the PELAN case, in that it is also recessive and probably encodes a protein with attenuated function.Similar lossof-function mutations resulting in decreased trichome formation have also been observed in cucumber, wherein the C2H2 transcription factor Tu is entirely absent, leading to the manifestation of the non-warty fruit trait [55].
By contrast, at the other nectar guide trichome loci, the M. parishii alleles are dominant and, based on the F1 phenotype (Fig. 1a, c, d), most likely encode repressors of trichome elongation.Although transgenic experiments showed that B-type cyclin-like and Jasmonate ZIM (JAZ) proteins can repress trichome initiation in systems other than Arabidopsis [51,[56][57][58], whether these repressors contribute to natural variation in trichome development is unclear.Interestingly, a recent study investigating natural trichome variation in vegetative tissues among snapdragon (Antirrhinum) species identified a glutaredoxin gene as a dominant repressor of trichome development [65].However, this glutaredoxin gene has acquired its trichome repressing function after the divergence between Antirrhinum and Mimulus [65], and thus is unlikely to cause trichome variation between our focal Mimulus species.Moreover, compared to loss-of-function mutations, the molecular mechanisms generating gain-of-function mutations are much less understood.Further characterization of the dominant repressor loci in M. parishii will not only be significant for the elucidation of negative regulators of trichome development, but also contribute to our understanding of the molecular basis of gain-offunction mutations during phenotypic evolution.

Fig. 1
Fig. 1 Phenotypes of nectar guide trichomes.(a) Front view of the whole corolla (upper) and enlarged view of the corolla throat of M. lewisii (Mlew, left), M. parishii (Mpar, middle), and their F1 hybrid (right).Scale bars, 5 mm (top row) and 1 mm (bottom row).The red circle marks the position where nectar guide trichome length were quantified.(b) Front view of F2 hybrids representing the four classes.Scale bars, 4 mm.(c) Light microscopy images of nectar guide trichomes of M. lewisii, M. parishii and F1 hybrids.Scale bars, 200 μm.(d) Quantification of nectar guide trichome length (n = 20 for each genotype).Error bars are 1 SD.Asterisks indicate differences from M. lewisii (** p < 0.01, student's t test).(e) Frequency distribution of the four classes of F2 individuals

Fig. 2
Fig. 2 Identification of the causal gene GUIDELESS.(a) Genome scan of the G P/P NIL for regions that are enriched in homozygous SNPs compared with the M. lewisii reference genome.(b) Cross design to generate a fine-scale mapping population (BC 3 S 1 ), and within this population, the three most informative recombinants reduced the candidate genomic interval to a smaller region (460 Kb) between markers MLCP6_200 and MLCP6_250.Nectar guide trichome length phenotypes (long vs. short) are shown on the right.(c) Front view of the whole corolla and enlarged view of the corolla throat of the G P/P NIL, the guideless mutant, F1 between guideless and the wild-type M. lewisii (GL_F1), and F1 between guideless and the G P/P NIL (GN_F1).Scale bars, 5 mm (top row) and 2 mm (bottom row).(d) Quantification of nectar guide trichome lengths (n = 20 for each genotype).Error bars are 1 SD.Asterisks indicate differences from the wild-type M. lewisii (** p < 0.01, student's t test).(e) Expression analysis of GUIDELESS by RT-qPCR

Fig. 3
Fig. 3 Multiple alignment of MIXTA-like R2R3-MYB protein sequences.Ml: Mimulus lewisii; Mp: Mimulus parishii; Am: Antirrhinum majus; Ph: Petunia hybrida; At: Arabidopsis thaliana.The signature motif defining the MIXTA-like clade of R2R3-MYBs is marked by a black bar above the alignment.MpGUIDE-LESS has the two amino acids "SA" in the highly conserved signature motif replaced by "TT".The two black triangles show the positions of the two introns